CN109952093A - A kind of SOST antibody pharmaceutical compositions and application thereof - Google Patents

Abstract

A kind of pharmaceutical composition, it includes the SOST antibody or its antigen-binding fragment in Acetic acid-sodium acetate buffer.In addition to this, which can also contain sugar, nonionic surface active agent and other auxiliary materials.Described pharmaceutical composition presents good Antibody stability after storing the several months.

Description

A kind of SOST antibody pharmaceutical compositions and application thereof Technical field

The invention belongs to field of pharmaceutical preparations, and in particular to the purposes of a kind of pharmaceutical composition comprising SOST antibody and its antigen-binding fragment as well as drug.

Background technique

Osteoporosis (Osteoporosis, it OP) include postmenopausal osteoporosis (Postmenopausal Osteoporosis,) and senile osteoporosis PMO, it is a kind of characterized by Low BMD and bone micro-structure are degenerated, bone strength decline, bone brittleness is caused to increase, be prone to the systemic dysostosis disease of fracture.According to statistics, there are about 200,000,000 people's osteoporosises, disease incidence has leapt to common disease, the 7th of frequently-occurring disease in the whole world.The illness rate of China 60 years old or more old women osteoporosis is up to 60%, and male's illness rate is also in 40-50%.

Other than reinforcing moving, taking the traditional measures such as calcium tablet and vitamin D, universal prevention of the fractures knowledge, existing drug therapy is mainly limited to reduce bone resorption to prevent from fracturing.Anti- bone resorption class drug includes calcitonin, Diphosphonate, estrogen replacement agent and selective estrogen receptor modulators (SERMs) etc..These using Diphosphonate as the bone resorption inhibitor of representative although further bone-loss can be prevented, can not but rebuild the sclerotin being lost, and while inhibiting bone resorption, equally inhibition ostosis.Hormone medicine more has the risk for causing phlebothrombosis and inducing cardiovascular disease.Importantly, bone amount not only can be improved in bone anabolism medicine truly, it should be able to also be effectively improved bone micro-structure and promote ostosis, and this point is exactly at present not available for anti-bone resorption class drug.In past 15 year, the various medical measures for being intended to reduce risk of bone fracture enter clinical experimental stage through systematization research, and practical optional medication is still very deficient.So far, only parathyroid hormone (PTH) class drug is proved to that ostosis can be stimulated, and still, shortcomings are also obvious to all.For example, it for bone remodelling effect not persistently, for repair of fractured bones effect less, need daily skin note administration 1 year or more, and can only low dosage administration, expensive, validity period must not exceed 2 years and the safety issue of its exposure is alerted by the black surround of U.S. FDA etc..

Sclerostin (Sclerostin) is used for drug development as new biological targets, and principle is can to treat osteoporosis by adjusting osteoblast anabolic, which, which fills up, treats osteoporosis field blank by regulating and controlling bone metabolism mode.

Sclerostin is the glycoprotein of SOST gene expression secretion, and the structure feature of amino acid sequence is 190 residues and the loop domain with cysteine.It has proven to its expression mainly to carry out in osteocyte, and there was only the expression of very low degree in positions such as osteoblast, cartilage, liver, kidney, marrow, heart, pancreases.

Studies have shown that sclerostin is by combining LDL receptor LRP5/6 to inhibit the ostosis of Wnt signal transduction regulatory.Current several companies come into clinical III, II phase for the monoclonal antibody drug of target spot exploitation respectively.The clinical data of three companies proves that the target spot passes through the mechanism of action of regulation bone metabolism treatment osteoporosis and its safety and significant drug effect.Wherein, the clinical report for the anti-sclerostin antibodies Romosozumab that Amgen and UCB. S.A.'s joint are released shows that its safety and tolerance are good, and subject bone density is significantly improved compared with blank group.The medicine has been advanced to clinical three stages phase.Two monoclonal antibody medicines that Lilly and Novartis company releases respectively have been in the clinical the second stage of stage.The applicable disease of these antibody is osteoporosis/osteoporosis and bone injury/related bone condition treatment etc..Does not conflict it is noted that some researches show that, anti-sclerostin antibodies with traditional bisphosphonates treatment, have associated with may.

But its molecular weight of antibody drug is big, structure is complicated, is easy to degrade, polymerize or undesirable chemical modification occurs etc. and become unstable.In order to make antibody be suitable for being administered, and it is able to maintain stability in storage and then use process, plays better effect, the stabilization formulations research of antibody drug is particularly important.

Although You Duo company includes SOST antibody and pharmaceutical preparation in research and development at present, such as: WO2016145961, WO2012028683, WO2012135035.But for new SOST antibody, it is still necessary to develop drug (preparation) composition comprising SOST for being more suitable for administration.

Summary of the invention

The present invention provides a kind of pharmaceutical composition, it includes SOST antibody or its antigen-binding fragments, and buffer, the buffer is selected from acetate buffer, histidine buffering liquid, phosphate buffer or Succinate Buffer, preferably acetate buffer, more preferably Acetic acid-sodium acetate buffer.

In alternative embodiments, in pharmaceutical composition, the SOST antibody or its antigen-binding fragment concentration are (i.e., it is formulated in the concentration of SOST antibody or its antigen-binding fragment in buffer) it is about 1mg/ml to 120mg/ml, preferably about 60mg/ml to 120mg/ml, it is further preferably about 80 to 120mg/ml, more preferably about 90 to 110mg/ml, further preferably 80-100mg/ml.Non-limiting embodiment includes 90mg/ml, 91mg/ml, 92mg/ml, 93mg/ml, 94mg/ml, 95mg/ml, 96mg/ml, 97mg/ml, 98mg/ml, 99mg/ml, 100mg/ml, 101mg/ml, 102mg/ml, 103mg/ml, 104mg/ml, 105mg/ml, 106mg/ml, 107mg/ml, 108mg/ml, 109mg/ml, 110mg/ml, most preferably from about 100mg/ml.

In alternative embodiments, the concentration of buffer is about 5mM to 30mM, and preferably about 10mM to 20mM, non-limiting embodiment includes 10mM, 12mM, 14mM, 16mM, 18mM, 20mM, most preferably from about 10mM.

In alternative embodiments, the pH value of buffer described in pharmaceutical composition is about 4.8 to 5.5, and preferably about 5.0 to 5.5, it can also be about 5.0,5.1,5.2,5.3,5.4,5.5, most preferably from about 5.0 in non-limiting embodiment.After buffer pharmaceutical composition of the invention, the final pH value of resulting pharmaceutical composition is about 0.2-0.3 higher than ph value of buffer solution.Therefore, in alternative embodiments, the pH value of pharmaceutical composition of the present invention is about 4.8 to 5.5, and preferably about 5.0 to 5.5, also optional about 5.0,5.1,5.2,5.3,5.4,5.5, most preferably from about 5.2,5.3 in non-limiting embodiment.

Further, in alternative embodiments, pharmaceutical composition also includes sugar." sugar " of the invention includes conventional composition (CH ₂O) _nAnd its derivative, including monosaccharide, disaccharides, trisaccharide, polysaccharide, sugar alcohol, reducing sugar, nonreducing sugar etc..It can be selected from glucose, sucrose, trehalose, lactose, fructose, maltose, dextran, glycerol, erythritol, glycerine, arabite, sylitol, D-sorbite, mannitol, close inner disaccharides, melezitose, melitriose, manninotriose, stachyose, maltose, lactulose, maltulose, sorbierite, maltitol, lactitol, iso- maltulose etc..Preferred sugar is non-reducing disaccharide, more preferably trehalose or sucrose.

In alternative embodiments, sugared concentration is about 40mg/ml to 95mg/ml in pharmaceutical composition, preferably about 60mg/ml to 90mg/ml, non-limiting embodiment includes 60mg/ml, 65mg/ml, 70mg/ml, 75mg/ml, 80mg/ml, 85mg/ml, 90mg/ml, more preferably about 80mg/ml.

In alternative embodiments, pharmaceutical composition also includes surfactant.It can be selected from polysorbate20, polysorbate80, poloxalkol, Triton, dodecyl sodium sulfate, dodecyl sodium sulfate, octyl glucoside sodium, lauryl-, myristyl-, sub- oil base-, stearyl-sulfobetaines, lauryl-, myristyl-, sub- oil base-, stearyl-sarcosine, sub- oil base-, myristyl-, cetyl-glycine betaine, lauroyl aminocarbonyl propyl-, Ke's card amidopropyl-, sub- oleamidopropyl-, myristamide propyl-, palmityl aminocarbonyl propyl-, isostearoyl aminocarbonyl propyl-glycine betaine, myristamide propyl-, palmityl aminocarbonyl propyl-, isostearoyl aminocarbonyl propyl-dimethyl amine, methyl cocoa acyl group sodium, methyl oleyl taurate, polyethylene glycol, polypropylene glycol, the copolymer etc. of ethylene and propylene glycol.Preferred surfactant is polysorbate80 or polysorbate20, more preferably polysorbate80.

In alternative embodiments, the concentration of surfactant is about 0.02mg/ml to 0.8mg/ml in pharmaceutical composition, preferably about 0.3mg/ml to 0.6mg/ml, non-limiting embodiment includes 0.3mg/ml, 0.35mg/ml, 0.4mg/ml, 0.45mg/ml, 0.5mg/ml, 0.55mg/ml, 0.6mg/ml, more preferably about 0.4mg/ml.

In alternative embodiments, pharmaceutical composition also includes viscosity modifier.The viscosity modifier can be selected from calcium salt, sodium chloride, magnesium chloride, arginine hydrochloric acid, preferably calcium chloride, calcium acetate.

In alternative embodiments, the concentration of calcium salt is preferably about 4.5mM to 20mM in pharmaceutical composition, preferably about 4.5mM to 10mM, non-limiting embodiment includes 4.5mM, 5mM, 5.5mM, 6.0mM, 6.5mM, 7.0mM, 7.5mM, 8.0mM, 8.5mM, 9.0mM, 9.5mM, 10mM, more preferably about 4.5mM.

In alternative embodiments, described pharmaceutical composition includes:

(a) 1 to 120mg/ml SOST antibody or its antigen-binding fragment,

(b) 5 to 30mM acetate buffer,

(c) 40 to 95mg/ml trehalose,

(d) 0.02 to 0.8mg/ml polysorbate80,

(e) 4.5 to 20mM calcium salt, the pH of preferably described pharmaceutical composition are about 4.8 to 5.5, and more preferably about 5.0 to 5.2.

In alternative embodiments, described pharmaceutical composition includes:

(a) 90 to 110mg/ml SOST antibody or its antigen-binding fragment,

(b) 10 to 20mM acetate buffer,

(c) 60 to 90mg/ml trehalose,

(d) 0.3 to 0.6mg/ml polysorbate80,

(e) 4.5 to 10mM calcium salt, the preferably pH of described pharmaceutical composition are 5.0 to 5.2.

In alternative embodiments, described pharmaceutical composition includes:

(a) the SOST antibody or its antigen-binding fragment of 100mg/ml,

(b) sodium-acetate buffer of 10mM,

(c) trehalose of 80mg/ml,

(d) polysorbate80 of 0.4mg/ml,

(e) calcium salt of 4.5mM, the preferably pH of described pharmaceutical composition are 5.0, and the acetate buffer is Acetic acid-sodium acetate buffer.

In alternative embodiments, described pharmaceutical composition includes:

(a) 80 to 100mg/ml SOST antibody or its antigen-binding fragment, and

(b) 10 to 20mM acetate buffer, and the pH of described pharmaceutical composition is 5.0 to 5.2.

In alternative embodiments, described pharmaceutical composition includes:

(a) the SOST antibody or its antigen-binding fragment of 100mg/ml,

(b) the Acetic acid-sodium acetate buffer of 10mM, pH 5.0

(c) trehalose of 80mg/ml,

(d) polysorbate80 of 0.4mg/ml,

(e) calcium chloride of 4.5mM.

In alternative embodiments, SOST antibody described in pharmaceutical composition or antigen-binding fragment have HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:3, SEQ ID NO:9 and SEQ ID NO:5 respectively, and

LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8 respectively.

In alternative embodiments, the SOST antibody or its antigen-binding fragment have the heavy chain variable region as shown in SEQ ID NO:10, and the light chain variable region as shown in SEQ ID NO:13.

In alternative embodiments, antibody described in pharmaceutical composition or antigen-binding fragment can be selected from source of mouse antibody, chimeric antibody, humanized antibody, preferably humanized antibody.

In alternative embodiments, the light chain of SOST antibody described in pharmaceutical composition and the antibody light chain amino acid sequence of Ab-5 have at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, the heavy chain amino acid sequence of the SOST antibody and the heavy chain of antibody of Ab-5 have at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, the Ab-5 antibody light chain sequences are as shown in SEQ ID NO:25, Ab-5 antibody heavy chain sequence As shown in SEQ ID NO:22.

The present invention also provides a kind of methods of lyophilized preparation for preparing the antibody containing SOST, including the step that foregoing pharmaceutical composition is freeze-dried.

In alternative embodiments, pre-freeze, primary drying and the redrying included the steps that successively is freeze-dried described in the method for preparing the lyophilized preparation of the antibody containing SOST.The pre-freeze is to freeze from 5 DEG C to -40 DEG C to -50 DEG C, and optimal is -45 DEG C, is not required to vacuum degree.The primary drying temperature is -30 DEG C to 0 DEG C, and optimal is -27 DEG C；Vacuum degree 0.05mBar-0.2mBar, optimal is 0.1mBar.20 DEG C -30 DEG C of redrying temperature, optimal is 25 DEG C, and for vacuum degree from 0.05mBar-0.2mBar, optimal is 0.1mBar, is down to vacuum degree 0.005mBar-0.02mBar, optimal is 0.01mBar.

The present invention also provides the lyophilized preparations that a kind of method of lyophilized preparation through aforementioned preparation antibody containing SOST prepares the resulting antibody containing SOST.

In some embodiments, the lyophilized preparation is in 2-8 DEG C of stable at least three moon, at least six moon, at least 12 months, and at least 18 months or at least 24 months.In some embodiments, which stablizes at least 7 days, at least 14 days or at least 28 days in 40 DEG C.

The present invention also provides the methods of the redissolution solution of lyophilized preparation of the preparation containing SOST antibody to redissolve solution used and be selected from but not limited to water for injection, physiological saline or glucose solution including the step of by aforementioned lyophilized preparation through redissolving.Redissolution of the present invention, which refers to, is dissolved in the protein formulation of lyophilized in diluent (selected from but not limited to water for injection, physiological saline or glucose solution), so that the albumen is dispersed in the preparation of reconstruct, corresponding redissolution solution can be obtained.

The redissolution solution of lyophilized preparation containing SOST antibody obtained by the method preparation for redissolving solution through the aforementioned lyophilized preparation containing SOST antibody that the present invention also provides a kind of.

In alternative embodiments, the redissolution solution containing SOST antibody, wherein the SOST antibody or its antigen-binding fragment concentration are 80mg/ml to 100mg/ml；Most preferably 100mg/ml.

In alternative embodiments, the redissolution solution containing SOST antibody, wherein the pH for redissolving solution is 4.8 to 5.5, preferably 5.3.

In alternative embodiments, the redissolution solution containing SOST antibody, wherein further including Acetic acid-sodium acetate buffer, the Acetic acid-sodium acetate buffer concentration is 10mM to 30mM, preferably 10mM.

In alternative embodiments, the redissolution solution containing SOST antibody, wherein further including disaccharides, the disaccharides is selected from trehalose or sucrose.

In alternative embodiments, the redissolution solution containing SOST antibody, wherein the disaccharides concentration is 45mg/ml to 90mg/ml, preferably 75mg/ml to 80mg/ml, most preferably 80mg/ml.

In alternative embodiments, the redissolution solution containing SOST antibody, wherein further including surfactant, the surfactant is polysorbate, preferably polysorbate80.

In alternative embodiments, the redissolution solution containing SOST antibody, wherein the concentration of surfactant is 0.3mg/ml to 0.6mg/ml, preferably 0.4mg/ml.

In alternative embodiments, the redissolution solution containing SOST antibody, wherein further including viscosity modifier, wherein the viscosity modifier is selected from calcium salt, sodium chloride, magnesium chloride or arginine monohydrochloride, it is preferable that the calcium salt is selected from calcium chloride or calcium acetate.

In alternative embodiments, the redissolution solution containing SOST antibody, wherein the concentration of the calcium salt is 4.5mM to 20mM, preferably 4.5mM to 10mM, most preferably 4.5mM.

The invention further relates to a kind of lyophilized preparation of antibody containing SOST, the lyophilized preparation can form pharmaceutical composition above-mentioned after redissolving.

The present invention further provides a kind of product or kits, and it includes the containers that any stable pharmaceutical composition described herein is housed.In some embodiments, which is neutral boron silica glass tubular injection bottle.

The present invention also provides the redissolution solution of pharmaceutical composition above-mentioned or lyophilized preparation or lyophilized preparation in preparation for treating the purposes in the drug for treating the relevant disease of SOST or illness, wherein the disease or illness are selected from the disease or obstacle of osteoporosis, sclerotin reduction or osteoarthritis, rheumatoid arthritis, periodontosis or Huppert's disease, preferably osteoporosis.

The present invention also provides a kind of relevant disease for the treatment of and prevention SOST or the methods of illness, including giving the pharmaceutical composition above-mentioned of required bacterium or the redissolution solution of lyophilized preparation or lyophilized preparation, wherein the disease or illness are selected from the disease or obstacle of osteoporosis, sclerotin reduction or osteoarthritis, rheumatoid arthritis, periodontosis or Huppert's disease, preferably osteoporosis.

The present invention also provides a kind of products comprising container, the redissolution solution in the container equipped with pharmaceutical composition above-mentioned or lyophilized preparation or lyophilized preparation.

It is appreciated that one of each embodiment described herein, some or all of characteristics can be combined to form other embodiments of the present invention.These and other aspects of the invention is apparent to those skilled in the art.These and other embodiment of the invention is further described by following detailed description.

Specific embodiment

One, term

In order to be easier to understand the present invention, certain technical and scientific terms are defined in detail below.Unless separately explicitly defining herein, all other technical and scientific term used herein all has the normally understood meaning of those skilled in the art of the art.

" buffer " refers to that the effect for being conjugated component by its Acid-Base is resistant to the buffer of pH variation.Example by buffer of the pH control in proper range includes acetate, succinate, gluconate, histidine, oxalates, lactate, phosphate, citrate, tartrate, fumarate, glycylglycine and other organic acid buffer liquids.

" histidine buffering liquid " is the buffer comprising histidine ion.The example of histidine buffering liquid includes histidine-hydrochloride, histidine-acetate, histidine-phosphate, the buffers such as histidine-sulfate, preferably histidine-hydrochloric acid salt buffer.Histidine-hydrochloric acid salt buffer is that histidine is formulated with hydrochloric acid or histidine with histidine hydrochloride.

" citrate buffer " is the buffer for including citrate ion.The example of citrate buffer includes citric acid-sodium citrate, citric acid-potassium citrate, citric acid-calcium citrate, citric acid-magnesium citrate etc..Preferred citrate buffer is citric acid-sodium citrate buffer solution.

" Succinate Buffer " is the buffer for including amber acid ion.The example of Succinate Buffer includes succinic acid-sodium succinate, succinic acid-Potassium Suceinate, succinic acid-succinic acid calcium salt etc..Preferred Succinate Buffer is succinic acid-Na-succinate buffer.

" phosphate buffer " is the buffer for including phosphate ion.The example of phosphate buffer includes disodium hydrogen phosphate acid-sodium dihydrogen phosphate, disodium hydrogen phosphate acid-potassium dihydrogen phosphate etc..Preferred phosphate buffer is disodium hydrogen phosphate acid-phosphate sodium dihydrogen buffer solution.

" acetate buffer " is the buffer for including acetate ion.The example of acetate buffer includes Acetic acid-sodium acetate, acetic acid-histidine salt, acetic acid-potassium acetate, acetic acid-calcium acetate, acetic acid-magnesium acetate etc..Preferred acetate buffer is Acetic acid-sodium acetate buffer.

" viscosity modifier " is to adjust the viscosity of preparation and the medicinal material of routine that is added, signified viscosity modifier is primarily referred to as inorganic salts and amino-acid salt herein, preferably, inorganic salts are selected from sodium chloride, calcium chloride, magnesium chloride or calcium acetate, preferably, the amino-acid salt is selected from arginine monohydrochloride, histidine hydrochloride, glycine hydrochloride, histidine acetate etc..

" pharmaceutical composition " indicates the mixture containing one or more compounds described herein or its physiologically/pharmaceutical salt or pro-drug and other chemical constituents, the other components such as physiology/pharmaceutical carrier and excipient.The purpose of pharmaceutical composition is the administration promoted to organism, plays bioactivity in turn conducive to the absorption of active constituent.Herein, " pharmaceutical composition " and " preparation " be not mutually exclusive.

The solution form of heretofore described pharmaceutical composition, if solvent therein is water without specified otherwise.

" lyophilized preparation " indicates that the pharmaceutical composition or pharmaceutical solutions of liquid or solution form pass through the preparation obtained after vacuum freeze drying step or pharmaceutical composition.

Freeze-drying of the invention includes pre-freeze, primary drying, redrying.The purpose of pre-freeze is frozen product, obtains crystalline solid.Pre-freezing temperature and pre-freeze speed are two important technical parameters, pre-freezing temperature are set as -45 DEG C in the present invention, pre-freeze speed is set as 1 DEG C/min.Primary drying is also referred to as that trunk is dry, is the Main Stage of sample freeze-drying.Purpose is while removing ice in product, to keep shape of product, destruction of the reduction of minimum to product.If temperature and the vacuum degree selection of primary drying are improper, collapsing for product will lead to.Higher temperature and vacuum degree can accelerate that efficiency is lyophilized, but also will increase the risk that product collapses simultaneously.The temperature of primary drying of the present invention can be the temperature of this field routine, such as -30 DEG C -0 DEG C.Redrying is also referred to as parsing-desiccation, is by taking out ultimate vacuum (0.01mbar) and increasing (20-40 DEG C) of the temperature key step removed in product in conjunction with water.Since most of biological products are more sensitive to temperature, so low spot of the redrying temperature selection in temperature range, i.e., 25 DEG C.The container of the time of freeze-drying and refrigerator, lyophilized preparation dosage, lyophilized medication is related.The adjustment of this time is well-known to those skilled in the art.

Terms used herein " about " is index value within the scope of the acceptable error of the occurrence measured by persons skilled in the art, and the numerical part depends on how to measure or measures (i.e. the limit of measurement system).For example, " about " may imply that standard deviation in 1 or more than 1 in this field is carried out each time.Alternatively, " about " or " basically comprising " may imply that at most 20% range.In addition, the term may imply that at most 5 times of at most an order of magnitude or numerical value for especially for biology system or process.Unless otherwise stated, otherwise when occurrence occurs in the application and claim, the meaning of " about " or " basically comprising " is it should be assumed that within the scope of the acceptable error of the occurrence.

Pharmaceutical composition of the present invention can achieve the effect that a kind of stable: antibody therein substantially retains its physical stability and/or chemical stability and/or biological activity after storage, preferably, pharmaceutical composition substantially retains its physics and chemical stability and its biological activity after storage.Storage period is generally basede on the predetermined storage life of pharmaceutical composition to select.At present there are many analytical technology of measurement protein stability, the stability after selected temperature stores seclected time period can measure.

Stable pharmaceutical antibody formulation is the preparation that significant changes are not observed in the following cases: being for up to 2 years in refrigerated storage temperature (2-8 DEG C) preservation at least three moon, preferably 6 months, 1 year more preferable, and even more preferably.In addition, stable liquid preparation includes such liquid preparation: it shows desired feature after the period including 25 DEG C save including 1 month, 3 months, 6 months or save 1 month at 40 DEG C.The typical acceptable standard of stability is as follows: being measured by SEC-HPLC, usually no more than about 10%, preferably more than about 5% antibody monomer is degraded.By visual analysis, pharmaceutical antibody formulation is colourless, or is clarified to slightly milky.Concentration, pH and the osmolality of the preparation, which have, is no more than ± 10% variation.It is generally observed the truncation of no more than about 10%, preferably more than about 5%, is usually formed the aggregation of no more than about 10%, preferably more than about 5%.

If after visual inspection color and/or clarity, or it is measured by the scattering of UV light, size exclusion chromatography (SEC) and dynamic light scattering (DLS), antibody does not show that significant aggregation increases, precipitates and/or is denaturalized, then the antibody " physical stability for retaining it " in pharmaceutical preparation.The variation of protein conformation can be evaluated by fluorescent spectrometry (it determines albumen tertiary structure) and by FTIR spectrum method (it determines Protein secondary structure).

If antibody does not show significant chemical modification, the antibody " chemical stability for retaining it " in pharmaceutical preparation.Albumen by way of changing in detection and quantitative chemical, it can be estimated that chemical stability.The degradation process for often changing protein chemistry structure includes hydrolyzing or truncating (evaluating by such as the methods of size exclusion chromatography and SDS-PAGE), oxidation (is evaluated) by the methods of peptide mapping such as in conjunction with mass spectrography or MALDI/TOF/MS, desamidation (passes through such as ion-exchange chromatography, capillary isoelectric focusing, peptide mapping, the methods of different aspartic acid measurement is evaluated) and isomerization (by the different aspartate content of measurement, peptide mapping etc. is evaluated).

If bioactivity of the antibody in given time is the antibody " bioactivity for retaining it " in pharmaceutical preparation in the preset range of the bioactivity shown when preparing pharmaceutical preparation.The bioactivity of antibody can be determined for example by antigen binding measurement.

In the present invention, term " sclerostin " or " SOST " or " SOST albumen " or " Sclerostin " refer to sclerostin (sclerostin or SOST) gene expression product (protein), it is refered in particular in the present invention as non-, such as mouse SOST (m-SOST) or Macaca inus SOST (cyno-SOST), refer both to the SOST (h-SOST) of people.People used in the present invention, mouse, Macaca inus SOST pass through GenBank and obtain nucleotide sequence, such as NP_079513.1 provides people SOST protein sequence.

Amino acid three-letter codes used in the present invention and single letter code such as J.biol.chem, described in 243, p3558 (1968).

" antibody " of the present invention refers to immunoglobulin, is four peptide chain structures being formed by connecting by two identical heavy chains and two identical light chains by interchain disulfide bond.

In the present invention, antibody light chain of the present invention can further include constant region of light chain, and the constant region of light chain includes κ, λ chain or its variant of source of people or source of mouse.

In the present invention, heavy chain of antibody of the present invention can further include heavy chain constant region, and the heavy chain constant region includes IgG1, IgG2, IgG3, IgG4 or its variant of source of people or source of mouse.

Heavy chain of antibody and light chain are very big close to the sequence variation of about 110 amino acid of N-terminal, are variable region (area Fv)；Remaining amino acid sequence close to C-terminal is relatively stable, is constant region.The variable region skeleton area (FR) relatively conservative including 3 hypervariable regions (HVR) and 4 sequences.3 hypervariable regions determine the specificity of antibody, also known as complementarity-determining region (CDR).Every light chain variable region (LCVR) and heavy chain variable region (HCVR) are by 3 CDR regions, 4 FR district's groups at the sequence being arranged successively from aminoterminal to c-terminus are as follows: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.3 CDR regions of light chain refer to LCDR1, LCDR2 and LCDR3；3 CDR regions of heavy chain refer to HCDR1, HCDR2 and HCDR3.The area LCVR of antibody or antigen-binding fragment of the present invention and the cdr amino acid residue in the area HCVR meet known Kabat coding rule (LCDR1-3 in quantity and position, HCDR2-3), or meet the coding rule (HCDR1) of kabat and chothia.

Antibody of the invention includes source of mouse antibody, chimeric antibody, humanized antibody, preferably humanized antibody.

Term " source of mouse antibody " is the monoclonal antibody to people SOST prepared according to this field knowledge and skills in the present invention.With SOST antigen injection subjects when preparation, then separation expression has the hybridoma of the antibody of required sequence or functional characteristic.

Term " chimeric antibody (chimeric antibody) " is that antibody made of the constant domain of the variable region of murine antibody and human antibody can be mitigated the immune response of murine antibody induction.Establish chimeric antibody, first to establish the hybridoma of secretion mouse specific monoclonal antibody, then variable region gene is cloned from mouse hybridoma cell, further according to the constant region gene for needing to clone human antibody, it is inserted into people's carrier after mouse variable region gene and human constant region gene are connected into mosaic gene, finally the chimeric antibody expression molecule in eukaryon industrial system or protokaryon industrial system.In a preferred embodiment of the present invention, the antibody light chain of the SOST chimeric antibody further includes source of people κ, λ chain or the constant region of light chain of its variant.The heavy chain of antibody of the SOST chimeric antibody further includes the heavy chain constant region of humanized IgG 1, IgG2, IgG3, IgG4 or its variant.

Term " humanized antibody (humanized antibody) ", also referred to as CDR grafted antibody (CDR-grafted antibody), refer to the antibody variable region frame that the CDR sequence of mouse is transplanted to people, i.e., the antibody generated in different types of human germline antibody's frame sequence.Chimeric antibody can be overcome due to carrying a large amount of murine protein ingredients, thus the strong antibody variable antibody response of induction.Such frame sequence can be obtained from the public DNA database or disclosed bibliography for including germline antibody gene sequences.As people's heavy chain and the germline DNA sequence dna of light-chain variable region gene can be in " VBase " human germ line sequences' databases (in internet www.mrccpe.com.ac.uk/vbaseCan get), and in Kabat, E.A. et al., 1991Sequences of Proteins of Immunological Interest is found in the 5th edition.While to avoid immunogenicity from declining, caused activity decline can carry out minimum inverse transition or back mutation to the human antibody variable region framework sequence, to keep activity.Humanized antibody of the invention also includes further carrying out the humanized antibody after affinity maturation to CDR by phage display.

Term " anti-sclerostin antibodies " in the present invention, " antibody of specific binding people's sclerostin ", " anti-SOST antibody ", " anti-SOST ", " SOST antibody " refer to such antibody " in conjunction with the antibody of SOST ", and the antibody can be with enough affinity combination SOST antibody so that the antibody may be used as the diagnosticum and/or therapeutic agent in targeting SOST.

Term " in conjunction with SOST " in the present invention, referring to can interact with people SOST.

Term " specific binding " refers to as by the available technology in this field, such as competitive ELISA, Measurement or It is measured.The term apply also for ought antibody for example of the present invention the special situation of antigen-binding domains defined epitope that many antigens are carried, the antibody for carrying antigen-binding domains in this case can specifically bind a variety of antigens for carrying the epitope.

Heretofore described " antigen-binding fragment ", refer to the Fab segment with antigen-binding activity, Fab ' segment, 2 segment of F (ab '), and scFv segment in conjunction with people SOST and other the segment for the combinable people SOST that can be formed with the area VH and VL of the antibody in conjunction with people SOST is utilized；It includes one or more CDR regions in SEQ ID NO:3-9 of antibody of the present invention.Fv segment contains antibody heavy chain variable region and light chain variable region, but does not have constant region, and has the minimum antibody fragment of whole antigen binding sites.Generally, Fv antibody also includes the peptide linker between VH and VL structural domain, and structure needed for being capable of forming antigen binding.Two antibody variable regions can also be connected into a polypeptide chain, referred to as single-chain antibody (single chain antibody) or scFv (sFv) with different attachments.

Term " epitope " refers in the antigen binding domain of one or more antibody can be by antibody identifies and combines molecular moiety.

" conservative modification " or " conservative substitution or substitution " refer to the amino acid in other amino acid replacement albumen with similar characteristics (such as charge, side chain size, hydrophobicity/hydrophily, Conformation of the main chain and rigidity etc.), so that can biological activity of the frequent progress change without changing albumen.As known to those skilled in the art, in general, single amino acids displacement in the non-essential region of polypeptide not substantially changes biological activity (see, for example, Watson etc. (1987) Molecular Biology of the Gene, The Benjamin/Cummings Pub.Co., page 224, (the 4th edition)).In addition, the unlikely broken ring biological activity of displacement of the similar amino acid of structure or function.

Amino acid sequence " identity " refers to two sequence similarities between albumen or polypeptide.When the position in two comparison sequences is occupied by same amino acid residue, for example, if when a position of two polypeptides is all occupied by the same amino acid residue, then the molecule is consistent in the position.The example for being adapted to determine that the algorithm of Percentage of sequence identity and sequence similarity percentage is BLAST and BLAST2.0 algorithm, they are described in Altschul et al. (1990) J.Mol.Biol.215:403-410 and Altschul et al. (1977) Nucleic Acids Res.25:3389-3402.Software for executing BLAST analysis can disclose acquisition (http://www.ncbi.nlm.nih.gov/) in National Center for Biotechnology Information.

The method of known production and antibody purification and antigen-binding fragment in the prior art, such as the antibody assay technical manual of Cold SpringHarbor, 5-8 chapter and 15 chapters.The CDR region that the antibody or antigen-binding fragment gene engineering method are invented in non-source of people adds the one or more area source of people FR.People FR Germline sequences can be by comparing the human antibody variable domains IMGT germ line genes database and MOE software, it is obtained from the website http://imgt.cines.fr of ImMunoGeneTics (IMGT), or it is obtained from immunoglobulin magazine, 2001ISBN012441351.

The antibody or antigen-binding fragment that the present invention is engineered can be prepared and purified with conventional method.For example, the cDNA sequence of encoding heavy chain and light chain, can clone and recombinate to GS expression vector.The immunoglobulin expression carrier of recombination can steadily transfection CHO cell.As the prior art that one kind is more recommended, mammal expression system will lead to the glycosylation of antibody, the especially highly conserved N-terminal site in the area Fc.Stable clone is obtained with the people SOST antibody specifically bound by expressing.Positive being cloned in the serum free medium of bioreactor expands culture to produce antibody.The culture solution for having secreted antibody can be purified with routine techniques.For example, being purified with A the or G Sepharose FF column containing adjusted buffer.Wash away the component of non-specific binding.The antibody for using pH gradient method elution of bound again detects antibody fragment with SDS-PAGE, collects.Antibody can be filtered concentration with conventional method.Soluble mixture and polymer can also be removed with conventional method, such as molecular sieve, ion exchange.Obtained product need to freeze immediately, and such as -70 DEG C, or freeze-drying.

" giving " and " processing " refers to the contact of exogenous drugs, therapeutic agent, diagnosticum or composition with animal, people, subject, cell, tissue, organ or biofluid when being applied to animal, people, experimental subjects, cell, tissue, organ or biofluid." giving " and " processing " can refer to such as treatment, pharmacokinetics, diagnosis, research and experimental method.The processing of cell includes contact and reagent contact with fluid of the reagent with cell, wherein the fluid is contacted with cell." giving " and " processing " still means that through reagent, diagnosis, combining compositions or passes through another cells in vitro and ex vivo treatment such as cell." processing " refers to treatment processing, prevention or preventive measure, research and diagnostic application when being applied to people, animal medicine or study subject.

" treatment " means to give the interior or topical therapeutic agent of patient, such as the composition comprising any binding compounds of the invention, and the patient has one or more disease symptoms, and the known therapeutic agent has therapeutic effect to these symptoms.In general, therapeutic agent is given in subject or group with the amount that one or more disease symptoms are effectively relieved, to induce this kind of symptom to degenerate or inhibit this kind of symptom development to any measurable degree of clinic.The amount (also referred to as " therapeutically effective amount ") that the therapeutic agent of any disease specific symptom is effectively relieved can change according to many factors, such as morbid state, age and the weight and drug of patient generate the ability for needing curative effect in patient.It is commonly evaluated for the seriousness of the symptom or any clinical testing procedure of development situation by doctor or other professional health care personages, whether evaluable disease symptoms have been mitigated.Although embodiment of the present invention (such as treatment method or product) may be invalid in terms of alleviating each target disease symptom, but examine (H inspection), Jonckheere-Terpstra inspection and Wilcoxon to examine and determine according to any statistical test method known in the art such as Student t inspection, Chi-square Test, according to the U inspection of Mann and Whitney, Kruskal-Wallis, target disease symptom should be mitigated in the patient of statistically significant number.

" effective quantity " includes to be enough to improve or the amount of the symptom of preventive medicine disease or illness.Effective quantity still means that the amount for being enough to allow or promoting diagnosis.It can change according to following factor for the effective quantity of particular patient or veterinary science subject: for example, illness to be treated, the general health of patient, the method and approach of administration and dosage and side effect seriousness.Effective quantity can be the maximum dose or dosage regimen for avoiding significant side effect or toxic effect.

" Tm value " refers to temperature when protein heat denaturation temperature, i.e. half albumen unfolding, and the space structure of albumen is destroyed at this time, so Tm value is higher, albumen thermal stability is higher.

Two, embodiment and test case

By following embodiment, present invention be described in more detail.These embodiment being merely to illustrate property purposes, and the range being not intended to restrict the invention.

Test method without specific conditions in the embodiment of the present invention, usually according to normal condition；Or according to condition proposed by raw material or commodity manufacturer.The reagent in specific source is not specified, for the conventional reagent of market purchase.

Embodiment

The preparation of SOST antigen and antibody, purification process are on 2 16th, 2016 in the applying date in the application, application No. is PCT/CN2016/073857, it is recorded in Publication No. WO2016145961 patent document, the full content of aforementioned application file can introduce the present invention.

Embodiment 1: anti-human SOST monoclonal antibody generates

Anti-human SOST monoclonal antibody is generated by immune mouse.The experiment white mouse of SJL, female, 6 week old (Beijing Vital River Experimental Animals Technology Co., Ltd., animal productiong licensing number: SCXK (capital) 2012-0001).Feeding environment: SPF grades.After mouse is bought, laboratory environment is raised 1 week, and light dark cycles are adjusted within 12/12 hour, and 20-25 DEG C of temperature；Humidity 40-60%.The mouse for having adapted to environment is divided into 2 groups (A/B), every group 10.

Immunizing antigen is the SOST recombinant protein (His-h-SOST) with His label.A group is emulsified with group with Freund's adjuvant (sigma Lot Num:F5881/F5506): first immunisation Freund's complete adjuvant (CFA), remaining booster immunization is with incomplete Freund's adjuvant (IFA).Antigen and adjuvant ratio are 1:1, and only (first immunisation), 200 μ l/100 μ g/ are only (booster immunization) by 200 μ l/200 μ g/.B group Titermax (sigma Lot Num:T2684) and Alum (Thremo Lot Num:77161) cross immunity.Antigen and adjuvant (titermax) ratio are 1:1, and antigen and adjuvant (Alum) ratio are 3:1, and only (first immunisation), 200 μ l/100 μ g/ are only (booster immunization) by 200 μ l/200 μ g/.It is inoculated with after antigen emulsification, the time is the 0th, 14,28,42,56 day.

Antigen after the emulsification of 50 μ g/ of (IP) injection only in 0th day A/B group peritonaeum.14th day subcutaneous 25 μ g/ of (sc) multiple spot (general back 6-8 point) injection is only.According to back agglomeration and abdomen swelling situation, back or intraperitoneal injection antigen are selected within 28th, 42 day.Preceding 3 days booster immunizations are merged carrying out splenocyte, the antigenic solution of the normal saline of 200 μ g/ of (IP) injection only in peritonaeum.

In progress blood examination in the 22nd, 36,50,64 day, the combination activity of mice serum and people's sclerostin was detected with the ELISA method of test case 1, the results are shown in Table 1.After the 4th is immune, selects Serum Antibody titre high and titre tend to the mouse of platform and carries out splenocyte fusion, the fusion steps mediated using the PEG of optimization by splenic lymphocytes and myeloma cell Sp2/0 cell ( CRL-8287 ^TM) merged to obtain hybridoma.And the activity that anti-SOST antibody blocking people SOST and people LRP-6 is combined in mice serum is detected by test case 2, the good monoclonal hybridoma strain Ab-1 of external activity is selected, Activity determination the results are shown in Table 1:

The external activity of 1. source of mouse antibody of table

Candidate antibodies	Test case 1-EC50 (nM)	Test case 2-IC50 (nM)
Ab-1	0.701	9.91

Embodiment 2: the humanization of the anti-human sclerostin antibodies of source of mouse

The good monoclonal hybridoma strain Ab-1 of external activity is picked out, clones monoclonal antibody sequences therein, then carry out humanization, recombinant expression and activity rating.

Cloned sequence process is as follows from hybridoma.Logarithmic growth phase hybridoma is collected, extracts RNA (according to kit specification step) with Trizol (Invitrogen, 15596-018), reverse transcription (PrimeScript ^TMReverse Transcriptase, Takara, cat#2680A).Sequencing company is sent to be sequenced after the cDNA that reverse transcription obtains is carried out PCR amplification using mouse Ig-Primer Set (Novagen, TB326Rev.B0503).Shown in the corresponding amino acid sequence SEQ ID NO:1 and SEQ ID NO:2 of obtained DNA sequence dna:

Hybridoma obtains heavy chain variable region:

Hybridoma obtains light chain variable region:

The method of the anti-human many document publicities in sclerostin monoclonal antibody source of people such as this field of source of mouse carries out.In short, user's constant domain substitutes parent (source of mouse antibody) constant domain, ethnic group antibody sequence is selected according to the homology of source of mouse antibody and human antibody, carries out humanization.The candidate molecules that the present invention selects activity good carry out humanization, as a result as follows.

1, the CDR region of source of mouse anti-sclerostin antibodies

The amino acid residue of VH/VLCDR is determined and is annotated by Kabat numbering system.

The CDR sequence of source of mouse Ab-1 is as described in Table 2 in the present invention:

The CDR sequence of 2. source of mouse anti-sclerostin antibodies of table

Antibody	Ab-1
Heavy chain CDR1	DYNLD(SEQ ID NO:3)
Heavy chain CDR2	DIDPNNGDILYNQKFRD(SEQ ID NO:4)
Heavy chain CDR3	RWAYYFDY(SEQ ID NO:5)
Light chain CDR1	KASQSVSNDVA(SEQ ID NO:6)
Light chain CDR2	YTSNRFT(SEQ ID NO:7)
Light chain CDR3	QQDYSSPVT(SEQ ID NO:8)

2, ethnic group system FR region sequence is selected

On the basis of source of mouse antibody VH/VLCDR typical structure obtained, by weight, light-chain variable sequence compared with antibody Germline database, the high ethnic group system template of homology is obtained.Wherein human germline light chain framework region comes from human kappa light chain gene, the preferred people's germline light chain template IGKV1-39*01 or IGKV4-1*01 of antibody of the present invention.Human germline heavy chain framework regions come from people's heavy chain, the preferred human germline heavy's template IGHV1-18*01 of antibody of the present invention.The CDR region of source of mouse antibody A b-1 is transplanted in the humanization template of selection, replaces humanization variable region, then recombinate with IgG constant region.Then, based on the three-dimensional structure of source of mouse antibody, there is the residue of direct interaction to embedding residue, with CDR region, and back mutation is carried out to the residue that the conformation of VL and VH has a major impact, and the optimization of CDR region chemically unstable amino acid residue (optimizes heavy chain CDR2, obtain new heavy chain CDR2 sequence: DIDPNDGDILYNQKFRD, SEQ ID NO:9), obtain final humanized molecule.Its weight chain variabl area sequence can be combined as shown in SEQ ID NO:10-12 with the optionally heavy chain constant region sequence shown in the SEQ ID NO:16-18；Light-chain variable sequence is combined with the constant light chain sequences as shown in SEQ ID NO:19 respectively as shown in SEQ ID NO:13-15.

1) heavy chain variable region:

The heavy chain variable region of Ab-5:

The heavy chain variable region of Ab-9:

The heavy chain variable region of Ab-10:

2) heavy chain constant region of each antibody is optionally from following sequence:

The heavy chain constant region (scarce K) of human IgG 4:

The heavy chain constant region of human IgG 4:

The heavy chain constant region of human IgG2:

3) light chain variable region:

The light chain variable region of Ab-5:

The light chain variable region of Ab-9:

The light chain variable region of Ab-10:

4) constant region of light chain comes from source of people κ chain:

It cloned respectively with gene cloning, the method for recombinant expression, express, purify above-mentioned antibody, the methods of combined ELISA, antigen receptor combination blocking experiment, Biacore, cytoactive detection finally select activity and keep best source antibody A b-5, Ab-9, Ab-10, sequence are as follows:

Ab-10 humanized antibody:

Heavy chain:

Light chain:

Ab-9 humanized antibody:

Heavy chain:

Light chain:

Ab-5 humanized antibody:

Heavy chain:

Remarks: the K of Ab-5 humanised antibody heavy chain's sequence end is possible to removed during antibody expression, therefore the heavy chain end in final products can have K, can also not have K, but this has no effect on the performance of product itself.

Light chain:

Exemplary antibodies pharmaceutical composition (preparation) preparation process

Step 1: SOST antibody (such as Ab-5) and stabilization formulations are configured to the preparation stoste of the antibody containing SOST, middle control sample detection is sterile after filtering.Stoste is crossed into 0.22 μm of PVDF filter core, collects filtrate.

Step 2: adjust loading amount to 1.1ml, jumped a queue in 2ml cillin bottle by filtrate is filling, respectively at it is filling start, it is filling it is intermediate, filling at the end of control detection content uniformity in sampling.

Step 3: opening Cover-rolling machine, adds aluminium lid, carry out rolling lid.

Step 4: visual inspection, confirms the defects of product is not allowed without loading amount.XiLin bottle label is pasted in printing；Printing paper cassette label, lock carton, mounted box, paster box label.

Embodiment 3: the screening of buffer system

Using following buffer, the SOST antibody A b-5 preparation that protein concentration is 80mg/mL is prepared:

1) 10mM Acetic acid-sodium acetate, pH5.0

2) 10mM histidine-acetic acid, pH5.0

3) 10mM histidine-acetic acid, pH5.5

4) 10mM disodium hydrogen phosphate-citric acid, pH5.5

5) 10mM disodium hydrogen phosphate-citric acid, pH7.0

6) 10mM disodium hydrogen phosphate-citric acid, pH7.5

When SOST antibody A b-5 is replaced into pH7.0 and 7.5 buffers, precipitating completely occurs for albumen.In conjunction with its isoelectric point scope, show that pH of buffer should be no more than 5.5.

It replaces successful preparation to be packed into 2mL cillin bottle with 1.5mL/ bottles, is shaken (25 DEG C, 300rpm) STABILITY MONITORING after being sealed with overlay film chlorinated butyl rubber bung.After shaking 3 days, disodium hydrogen phosphate-citric acid (pH5.5) system is also completely muddy.Milky is presented in histidine-acetate system appearance after shaking, and a small amount of precipitating occurs in acetate system, and the two SEC purity is without significant changes.Therefore SOST antibody preparation buffer system can be histidine-acetate system or Acetic acid-sodium acetate system, pH range should be 5.0-5.5.Wherein, Acetic acid-sodium acetate (pH5.0) is relatively better buffer system.It is shown in Table 3:

Table 3.Ab-5 shakes 3 days stability in different pH and buffer system

Use the SOST antibody preparation that in following buffer, preparation protein concentration is 85mg/mL:

1) 10mM Acetic acid-sodium acetate, pH4.8

2) 10mM Acetic acid-sodium acetate, pH5.0

3) 10mM Acetic acid-sodium acetate, pH5.2

4) 30mM Acetic acid-sodium acetate, pH5.0

5) 10mM succinic acid-sodium succinate, pH5.0

Preparation is packed into 2mL cillin bottle with 1.5mL/ bottles, is shaken (25 DEG C, 300rpm) stability and illumination (4500Lx) STABILITY MONITORING after being sealed with overlay film chlorinated butyl rubber bung.After shaking 3 days, muddiness completely occurs for 10mM Acetic acid-sodium acetate (pH4.8) system and 10mM succinic acid-sodium succinate (pH5.0) system.10mM Acetic acid-sodium acetate (pH5.0) and 30mM Acetic acid-sodium acetate (pH5.0) system are relatively preferable in shaking rear stability, and the two is without marked difference.After illumination 10 days, for SOST antibody in 10mM Acetic acid-sodium acetate (pH5.0) and 10mM Acetic acid-sodium acetate (pH5.2) system, the more other three groups of decline of IEC main peak purity is less.Therefore, the optimal buffer system of SOST antibody preparation is Acetic acid-sodium acetate system, and concentration can be 10-30mM, and preferably 10mM, pH should be greater than 4.8.

Table 4.Ab-5 shakes 3 days stability in different pH, ionic strength and buffer system

Table 5.Ab-5 10 days stability of illumination in different pH, ionic strength and buffer system

Embodiment 4: sugared screening in preparation

In 10mM succinic acid-sodium succinate, pH5.5 buffer, preparation Ab-5 protein concentration is 1mg/ml, 4.5%, 6%, 7.5%, 9% α contained respectively, the SOST antibody-solutions of α-Trehalose Dihydrate or 6% sucrose.With thermal stability of differential scanning calorimetry (differential scanning calorimetry, DSC) measurement SOST antibody in different prescriptions.The results show that with the increase of sugared concentration, the initial denaturation temperature (Tm of SOST antibody A b-5 _onset) be gradually increasing；And the Tm in trehalose _onsetIt is apparently higher than sucrose.Imply better heat stability of the SOST antibody in relatively high trehalose concentration.

Requirement in view of injection to osmotic pressure, selects 8% α, and α-Trehalose Dihydrate should be most preferably condition.

Thermal stability of the table 6.Ab-5 in different sugar types and concentration

Note: sugared concentration 6% is 60mg/ml in the present invention；9% is 90mg/ml.Other unit conversions and so on.

Embodiment 5: the screening of preparation medium viscosity regulator

In order to reduce formulation viscosity, containing 30mg/ml SOST antibody, 10mM Acetic acid-sodium acetate in pH5.0 solution, is separately added into following viscosity modifier, and can investigation reduce viscosity, improve ultrafiltration efficiency:

1) 90mg/ml α, α-Trehalose Dihydrate

2) 70mg/ml α, α-Trehalose Dihydrate+40mM sodium chloride

3) 90mg/ml α, α-Trehalose Dihydrate+10mM calcium chloride

4) 90mg/ml α, α-Trehalose Dihydrate+10mM magnesium chloride

5) 150mM sodium chloride

6) 140mM arginine monohydrochloride

7) 10mM calcium acetate

8) 20mM calcium chloride

9) 4.5mM calcium chloride

The experimental results showed that sugar is not helped for improving ultrafiltration efficiency, sodium chloride, arginine monohydrochloride can moderately increase ultrafiltration efficiency, and calcium salt and magnesium salts have great improvement for ultrafiltration efficiency.Wherein, the concentration range of calcium salt can be 4.5-20mM, which filters efficiency without marked difference.

Embodiment 6: the screening of surfactant in preparation

In the buffer containing various concentration and the surfactant of type, different protein concentrations, Acetic acid-sodium acetate containing 10mM (pH5.0), 4.5mM calcium chloride, 80mg/mL α, α-Trehalose Dihydrate SOST antibody preparation are prepared.Experimental design is carried out by DoE and analysis, range are as follows:

1) protein concentration: 80~100mg/ml

2) kinds of surfactants: polysorbate 20 (PS20), polyoxyethylene sorbitan monoleate (PS80)

3) polysorbate concentration: 0.02~0.8mg/ml

It by preparation with 1.2ml/ branch, is placed in 1mlBD administration injection device, carries out shaking in 25 DEG C/300rpm/12 days.The results show that SEC, non-reduced CE-SDS and IEC detection purity of all prescriptions after shaking 12 days are without significant changes.Illustrate the excellent compatibility of SOST antibody preparation and drug delivery device.Meanwhile sub- visible particle number and its situation of change of the MFI detection greater than 2 μm or more, show that there were significant differences between each prescription.According to DoE fitting result (models fitting is effective), using polyoxyethylene sorbitan monoleate, protein concentration is higher than 96mg/ml, can control granule number incrementss within 500.On this basis, polyoxyethylene sorbitan monoleate concentration can make particle in prescription minimum at 0 in about 0.3~0.6mg/ml.

Shaking stability (purity) of the Ab-5 of 7. various concentration of table in different tween types and concentration

Shaking stability (SVP) of the Ab-5 of 8. various concentration of table in different tween types and concentration

Embodiment 7: the stability study of formulation ingredients

Ab-5 is prepared in 10mM Acetic acid-sodium acetate buffer (pH5.0), 4.5mM calcium chloride, 80mg/mL α, α-Trehalose Dihydrate or sucrose, 0.4mg/mL polysorbate 20 or 80 with 100mg/mL.Preparation is packed into 2mL cillin bottle with 1.5mL/ bottles, after the sealing of overlay film chlorinated butyl rubber bung, is respectively placed in 25 DEG C and 2~8 DEG C progress STABILITY MONITORINGs.The result shows that the prescription of polysorbate 20 a large amount of little particles occurs after placing 6 months at 2~8 DEG C, SOST antibody A b-5 preparation should not be used in.In the prescription of polyoxyethylene sorbitan monoleate, trehalose and sucrose are placed at 25 DEG C without significant difference and place within 3 months or 2~8 DEG C 6 and monthly show good stability.

Table 9.Ab-5 different sugar and tween prescription place 25 DEG C of stability

Table 10.Ab-5 different sugar and tween prescription place 2~8 DEG C of stability

Embodiment 8: the freeze-drying of preparation

With the buffer of the Acetic acid-sodium acetate containing 10mM of pH5.0, preparation SOST antibody protein concentration is 100mg/ml, trehalose containing 80mg/ml, 0.4mg/ml polysorbate80, the anti-SOST antibody preparation of 4.5mM calcium chloride.Antibody is packed into 2mL cillin bottle with 1.1mL/ bottles, is fitted into freeze drying box, is lyophilized.Freeze-drying program is pre-freeze, primary drying and redrying.After EP (end of program) is lyophilized, vacuum is jumped a queue.It redissolves before and after sample is lyophilized and compares.The result shows that the good performance of liquid preparation can be kept by redissolving solution.

The step of freeze drying of 11. preparation of table

Embodiment 9: other optional pharmaceutical formulations

Stable pharmaceutical preparation provided by the invention, which can also be, appoints stable combination selected from the following:

(1) SOST antibody A b-5 120mg/ml, 95mg/ml trehalose, the polysorbate80 of 0.5mg/ml, the calcium chloride of 5mM Acetic acid-sodium acetate buffer and 15mM, final pH 5.5；

(2) SOST antibody A b-5 120mg/ml, 95mg/ml trehalose, the polysorbate80 of 0.5mg/ml, the calcium chloride of 5mM Acetic acid-sodium acetate buffer and 15mM, final pH 5.4；

(3) SOST antibody A b-5 120mg/ml, 95mg/ml trehalose, the polysorbate80 of 0.5mg/ml, the calcium chloride of 5mM Acetic acid-sodium acetate buffer and 15mM, final pH 5.3；

(4) SOST antibody A b-5 120mg/ml, 95mg/ml trehalose, the polysorbate80 of 0.5mg/ml, the calcium chloride of 5mM Acetic acid-sodium acetate buffer and 15mM, final pH 5.1；

(5) SOST antibody A b-5 120mg/ml, 95mg/ml trehalose, the polysorbate80 of 0.5mg/ml, the calcium chloride of 5mM Acetic acid-sodium acetate buffer and 15mM, final pH 4.9；

(6) SOST antibody A b-5 120mg/ml, 95mg/ml trehalose, the polysorbate80 of 0.5mg/ml, the calcium chloride of 5mM Acetic acid-sodium acetate buffer and 15mM, final pH 4.8；

(7) SOST antibody A b-5 110mg/ml, 40mg/ml trehalose, the polysorbate80 of 0.5mg/ml, the calcium chloride of 20mM Acetic acid-sodium acetate buffer and 7.5mM, final pH 5.4；

(8) SOST antibody A b-5 110mg/ml, 40mg/ml trehalose, the polysorbate80 of 0.5mg/ml, the calcium chloride of 20mM Acetic acid-sodium acetate buffer and 7.5mM, final pH 5.3；

(9) SOST antibody A b-5 110mg/ml, 40mg/ml trehalose, the polysorbate80 of 0.5mg/ml, the calcium chloride of 20mM Acetic acid-sodium acetate buffer and 7.5mM, final pH 5.1；

(10) SOST antibody A b-5 110mg/ml, 40mg/ml trehalose, the polysorbate80 of 0.5mg/ml, the calcium chloride of 20mM Acetic acid-sodium acetate buffer and 7.5mM, final pH 4.9；

(11) SOST antibody A b-5 100mg/ml, 50mg/ml trehalose, the polysorbate80 of 0.5mg/ml, the calcium chloride of 20mM Acetic acid-sodium acetate buffer and 6.0mM, final pH 4.9；

(12) SOST antibody A b-5 100mg/ml, 50mg/ml trehalose, the polysorbate80 of 0.5mg/ml, the calcium chloride of 20mM Acetic acid-sodium acetate buffer and 6.0mM, final pH 5.1；

(13) SOST antibody A b-5 100mg/ml, 50mg/ml trehalose, the polysorbate80 of 0.5mg/ml, the calcium chloride of 20mM Acetic acid-sodium acetate buffer and 6.0mM, final pH 5.3；

(14) SOST antibody A b-5 100mg/ml, 50mg/ml trehalose, the polysorbate80 of 0.5mg/ml, the calcium chloride of 20mM Acetic acid-sodium acetate buffer and 6.0mM, final pH 5.4.

(15) SOST antibody A b-5 90mg/ml, 40mg/ml trehalose, the polysorbate80 of 0.3mg/ml, the calcium chloride of 5mM Acetic acid-sodium acetate buffer (its pH is 4.8) and 15mM；

(16) SOST antibody A b-5 110mg/ml, 95mg/ml trehalose, the polysorbate80 of 0.6mg/ml, the calcium chloride of 20mM Acetic acid-sodium acetate buffer (its pH is 5.5) and 20mM；

(17) SOST antibody A b-5 100mg/ml, 80mg/ml trehalose, the polysorbate80 of 0.4mg/ml, the calcium chloride of 10mM Acetic acid-sodium acetate buffer (its pH is 5.0) and 4.5mM.

Claims (34)

1. A kind of pharmaceutical composition, it includes SOST antibody or its antigen-binding fragment and buffer,

   The buffer is selected from acetate buffer, histidine buffering liquid, phosphate buffer or Succinate Buffer, preferably acetate buffer, more preferably Acetic acid-sodium acetate buffer,

   Wherein, in described pharmaceutical composition, the SOST antibody or its antigen-binding fragment concentration are 1mg/ml to 120mg/ml.
2. Pharmaceutical composition as described in claim 1, in described pharmaceutical composition, the SOST antibody or its antigen-binding fragment concentration are 60mg/ml to 120mg/ml, preferably 90mg/ml to 120mg/ml, more preferably 80mg/ml to 100mg/ml, most preferably 100mg/ml.
3. Pharmaceutical composition as claimed in claim 1 or 2, wherein the pH of described pharmaceutical composition is 4.8 to 5.5, preferably 5.0 to 5.2.
4. Pharmaceutical composition as claimed any one in claims 1 to 3, wherein the buffer concentration is 5mM to 30mM, preferably 10mM to 20mM, most preferably 10mM.
5. Pharmaceutical composition as claimed in claim 4, wherein the pH of the buffer is 4.8 to 5.5, preferably 5.0 to 5.5, more preferably 5.0 to 5.2, most preferably 5.0.
6. Pharmaceutical composition as described in any one of claims 1 to 5, wherein further including disaccharides, the disaccharides is selected from trehalose or sucrose.
7. Pharmaceutical composition as claimed in claim 6, wherein the disaccharides concentration is 40mg/ml to 95mg/ml, preferably 45mg/ml to 90mg/ml, more preferably 60mg/ml to 90mg/ml, most preferably 80mg/ml.
8. Pharmaceutical composition as described in any one of claims 1 to 7, wherein further including surfactant, the surfactant is polysorbate, preferably polysorbate80.
9. Pharmaceutical composition as claimed in claim 8, wherein the concentration of the surfactant is 0.02mg/ml to 0.8mg/ml, preferably 0.3mg/ml to 0.6mg/ml, most preferably 0.4mg/ml.
10. Pharmaceutical composition as claimed in any one of claims 1-9 wherein, wherein further including viscosity modifier, the viscosity modifier is selected from calcium salt, sodium chloride, magnesium chloride or arginine monohydrochloride,

    Preferably, the calcium salt is selected from calcium chloride or calcium acetate.
11. Pharmaceutical composition as claimed in claim 10, wherein the concentration of the calcium salt is 4.5mM to 20mM, preferably 4.5mM to 10mM, most preferably 4.5mM.
12. A kind of pharmaceutical composition, wherein including:

    (a) 1 to 120mg/ml SOST antibody or its antigen-binding fragment, (b) 5 to 30mM acetate buffer, (c) 45 to 90mg/ml trehalose, (d) 0.02 to 0.8mg/ml polysorbate80, (e) 4.5 to 20mM calcium salt, it is preferred that the pH of described pharmaceutical composition is 4.8 to 5.5, more preferably 5.0 to 5.2；

    Or, described pharmaceutical composition includes the SOST antibody or its antigen-binding fragment of (a) 80 to 100mg/ml, (b) 10 to 20mM acetate buffer, its pH is 4.8 to 5.5, (c) 60 to 90mg/ml trehalose, (d) 0.3 to 0.6mg/ml polysorbate80, (e) 4.5 to 10mM calcium salt.
13. A kind of pharmaceutical composition, it includes:

    (a) 1 to 120mg/ml SOST antibody or its antigen-binding fragment, and

    (b) 10 to 20mM acetate buffer, and the pH of described pharmaceutical composition is 5.0 to 5.2.
14. Pharmaceutical composition as described in any one of claims 1 to 13, wherein the SOST antibody or its antigen-binding fragment have HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:3, SEQ ID NO:9 and SEQ ID NO:5 respectively, and

    LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8 respectively.
15. Pharmaceutical composition as claimed in claim 14, wherein the SOST antibody or its antigen-binding fragment have the heavy chain variable region as shown in SEQ ID NO:10, and the light chain variable region as shown in SEQ ID NO:13.
16. Such as the described in any item pharmaceutical compositions of claim 1 to 13, wherein the light-chain amino acid sequence of the SOST antibody has at least 90% sequence identity with sequence shown in SEQ ID NO:25, and sequence shown in the heavy chain amino acid sequence and SEQ ID NO:22 of the SOST antibody has at least 90% sequence identity.
17. The method for preparing the lyophilized preparation of the antibody containing SOST, including the step that pharmaceutical composition described in any one of claims 1 to 16 is freeze-dried.
18. The method of the lyophilized preparation of preparation antibody containing SOST as claimed in claim 17, wherein the freeze-drying includes the steps that pre-freeze, primary drying and redrying successively.
19. A kind of method as described in claim 17 or 18 prepares the lyophilized preparation of the resulting antibody containing SOST.
20. The method of redissolution solution of the preparation containing SOST antibody, including the step of by lyophilized preparation described in claim 19 through redissolving, wherein redissolving solvent for use is preferably water for injection.
21. It is a kind of that the resulting redissolution solution containing SOST antibody is prepared by method of claim 20.
22. Redissolution solution containing SOST antibody as claimed in claim 21, wherein the SOST antibody or its antigen-binding fragment concentration are 80mg/ml to 100mg/ml；Most preferably 100mg/ml.
23. Redissolution solution containing SOST antibody as claimed in claim 21, wherein the pH for redissolving solution is 4.8 to 5.5, preferably 5.3.
24. Redissolution solution containing SOST antibody as claimed in claim 21, wherein further including Acetic acid-sodium acetate buffer, the Acetic acid-sodium acetate buffer concentration is 10mM to 30mM, preferably 10mM.
25. Redissolution solution containing SOST antibody as claimed in claim 21, wherein further including disaccharides, the disaccharides is selected from trehalose or sucrose.
26. Redissolution solution containing SOST antibody as claimed in claim 25, wherein the disaccharides concentration is 45mg/ml to 90mg/ml, preferably 75mg/ml to 80mg/ml, most preferably 80mg/ml.
27. Redissolution solution containing SOST antibody as claimed in claim 21, wherein further including surfactant, the surfactant is polysorbate, preferably polysorbate80.
28. Redissolution solution containing SOST antibody as claimed in claim 27, wherein the concentration of surfactant is 0.3mg/ml to 0.6mg/ml, preferably 0.4mg/ml.
29. Redissolution solution containing SOST antibody as claimed in claim 21, wherein further include viscosity modifier, wherein the viscosity modifier is selected from calcium salt, sodium chloride, magnesium chloride or arginine monohydrochloride,

    Preferably, the calcium salt is selected from calcium chloride or calcium acetate.
30. Redissolution solution containing SOST antibody as claimed in claim 29, wherein the concentration of the calcium salt is 4.5mM to 20mM, preferably 4.5mM to 10mM, most preferably 4.5mM.
31. A kind of lyophilized preparation of the antibody containing SOST, it is characterised in that the lyophilized preparation can form the described in any item pharmaceutical compositions of claim 1 to 16 after redissolving.
32. Redissolution solution described in any one of lyophilized preparation described in pharmaceutical composition described in any one of claims 1 to 16 or claim 19 or 31 or claim 21-30 is preparing the purposes in the drug for treating the relevant disease of SOST or illness,

    Wherein the relevant disease of SOST or illness are selected from the disease or obstacle of osteoporosis, sclerotin reduction or osteoarthritis, rheumatoid arthritis, periodontosis or Huppert's disease, preferably osteoporosis.
33. A method for the treatment of and preventing the relevant disease of SOST or illness, including giving the redissolution solution as described in any one of lyophilized preparation or claim 21-30 as described in the pharmaceutical composition or claim 19 or 31 as described in any one of claims 1 to 16 of required bacterium

    Wherein the relevant disease of SOST or illness are selected from the disease or obstacle of osteoporosis, sclerotin reduction or osteoarthritis, rheumatoid arthritis, periodontosis or Huppert's disease, preferably osteoporosis.
34. A kind of product comprising container, equipped with redissolution solution described in any one of lyophilized preparation or claim 21-30 described in pharmaceutical composition or claim 19 or 31 described in any one of claims 1 to 16 in the container.